Blotting method for rapidly analyzing nucleic acid

Abstract

The present invention relates to a blotting method for rapidly analyzing nucleic acid comprising the steps of transferring a nucleic acid to be analyzed to the substrate and fixing the nucleic acid to be analyzed absorbed on the substrate; directing adding a nucleic acid probe to hybridize in a short time, without blocking the areas where the nucleic acid to be analyzed has not been fixed; removing the nucleic acid probe which has not been annealed to the nucleic acid to be analyzed by washing; and finally detecting the hybridization signal. According to the present invention, since the prehybridization is not needed and the hybridization and washing time is shortened, the time for the nucleic acid hybridization is dramatically shortened. Therefore, the whole blotting procedures for rapidly analyzing nucleic acid may be finished quickly.

1. A blotting method without the process of blocking for analyzing nucleic acid comprising the following steps:
 (1) providing a substrate having a plurality of pores; 
 (2) transferring a nucleic acid to be analyzed to the substrate and allowing absorption of the nucleic acid by the substrate; 
 (3) fixing the nucleic acid to be analyzed on the substrate, and the substrate is a dry substrate; 
 (4) adding a solution containing a nucleic acid probe on the dry substrate of step (3) to base-pair the nucleic acid probe with the nucleic acid to be analyzed thereon for two or more minutes, without blocking the areas of the substrate where the nucleic acid to be analyzed has not been fixed; 
 (5) removing the nucleic acid probe which has not been annealed to the nucleic acid to be analyzed of step (4); and 
 (6) detecting the hybridization signal on the substrate having been subjected to step (5), wherein in step (3), the substrate is dried at a temperature of 80° C. to 130° C. to fix the nucleic acid to be analyzed on the substrate and the drying time is 1 to 10 minutes. 
 
     
     
       2. The blotting method for analyzing nucleic acid according to  claim 1 , wherein the substrate in step (1) is also dry. 
     
     
       3. The blotting method for analyzing nucleic acid according to  claim 1 , wherein the substrate in step (1) is a membrane. 
     
     
       4. The blotting method for analyzing nucleic acid according to  claim 3 , wherein the substrate is a nylon membrane or nitrocellulose membrane. 
     
     
       5. The blotting method for analyzing nucleic acid according to  claim 1 , wherein the pores of the substrate have a diameter of 0.1to 50 μm. 
     
     
       6. The blotting method for analyzing nucleic acid according to  claim 1 , wherein in step (3), the substrate is radiated with ultraviolet light to fix the nucleic acid to be analyzed on the substrate. 
     
     
       7. The blotting method for analyzing nucleic acid according to  claim 6 , wherein after step (3), the substrate having the nucleic acid to be analyzed fixed thereon is further dried. 
     
     
       8. The blotting method for analyzing nucleic acid according to  claim 1 , wherein in step (4), the base pairing time is 2 to 5 minutes. 
     
     
       9. The blotting method for analyzing nucleic acid according to  claim 1 , wherein in step (5), a buffer solution is used for washing off the nucleic acid probe that has not been annealed to the nucleic acid to be analyzed. 
     
     
       10. The blotting method for analyzing nucleic acid according to  claim 9 , wherein in step (5), the buffer solution having low ionic strength, wherein the buffer solution comprises a 0.05 to 0.15 fold standard sodium citrate. 
     
     
       11. The blotting method for analyzing nucleic acid according to  claim 10 , wherein in step (5), the buffer solution of low ionic strength further comprises a sodium dodecyl sulfate of 0.05% to 0.15%(w/v). 
     
     
       12. The blotting method for analyzing nucleic acid according to  claim 11 , wherein in step (5), for the buffer solution of low ionic strength, the washing time is 3 to 6 minutes. 
     
     
       13. A blotting method for analyzing nucleic acid comprising the following steps:
 (1) providing a dry substrate which has a plurality of pores and is dry; 
 (2) transferring a nucleic acid to be analyzed to the dry substrate and allowing absorption of the nucleic acid by the substrate without pre-wetting; 
 (3) fixing the nucleic acid to be analyzed on the substrate; 
 (4) interacting a solution containing a nucleic acid probe with the substrate of step (3) at a temperature of 40° C. to 70° C., to base-pair the nucleic acid probe with the nucleic acid to be analyzed thereon for two or more minutes; 
 (5) washing the substrate of step (4) with a buffer solution to remove the nucleic acid probe which has not been annealed to the nucleic acid to be analyzed; and 
 (6) detecting the hybridization signal on the substrate having been subjected to step (5). 
 
     
     
       14. A blotting method for analyzing nucleic acid comprising the following steps:
 (1) providing a substrate having a plurality of pores; 
 (2) transferring a nucleic acid probe to the substrate and allowing absorption of the nucleic acid probe by the substrate without pre-wetting; 
 (3) fixing the nucleic acid probe on the substrate, and then drying the substrate again into a dry substrate; 
 (4) interacting a solution containing a nucleic acid to be analyzed with the dry substrate of step (3) at a temperature of 40° C. to 70° C., to base-pair the nucleic acid probe with the nucleic acid to be analyzed thereon for two or more minutes; 
 (5) washing the substrate of step (4) with a buffer solution to remove the nucleic acid to be analyzed which has not been annealed to the nucleic acid probe; and 
 (6) detecting the hybridization signal on the substrate having been subjected to step (5). 
 
     
     
       15. A blotting method for analyzing nucleic acid comprising the following steps:
 (1) providing a substrate having a plurality of pores; 
 (2) transferring a nucleic acid to be analyzed to the substrate and allowing absorption of the nucleic acid by the substrate; 
 (3) fixing the nucleic acid to be analyzed on the substrate, and the substrate is dry; 
 (4) adding a solution containing a nucleic acid probe on the dry substrate of step (3) by using a pressure difference generated by a capillarity of the substrate, entering it in the substrate, and base pairing the nucleic acid probe with the nucleic acid to be analyzed thereon for two or more minutes; 
 (5) washing the substrate of step (4) with a buffer solution to remove the nucleic acid probe which has not been annealed to the nucleic acid to be analyzed; and 
 (6) detecting the hybridization signal on the substrate having been subjected to step (5). 
 
     
     
       16. The blotting method for analyzing nucleic acid according to  claim 15 , wherein in step (4), the pressure difference is resulted by vacuuming on a first side of the substrate to form a negative pressure to make the nucleic acid probe sucked into a second side of the substrate, or by pressuring to push the nucleic acid probe into at least one of the first and second sides of the substrate.